This invention relates to nucleic acid sequences corresponding to the human gene for type C Niemann-Pick disease. The sequences are useful, among other applications, for the diagnosis of this disease. Also provided is the mouse homolog of this human gene.
Niemann-Pick disease is the name given to a class of inherited lipid storage diseases. Four types of the disease are recognized, Types A, B, C and D. Niemann-Pick disease type C (NP-C) is an autosomal recessive neurovisceral lipid storage disorder which leads to systemic and neurological abnormalities (Brady et al., 1989). Clinical features of the disease include variable hepatosplenomegaly, vertical supra-nuclear ophthalmoplegia, progressive ataxia, dystonia and dementia (Pentchev et al., 1989). Cataplexy and seizures may occur later in the course of the illness. NP-C is characterized by phenotypic variability, with onset ranging from birth to early adulthood (Fink et al.. 1989). Type C Niemann-Pick disease differs from types A and B in that the latter two forms are lipidoses resulting from a lesion in the sphingomyelinase gene located on chromosome 11 (Pereira et al., 1991). In contrast, the underlying genetic defect of NP-C remains unknown. Type D Niemann-Pick disease (also known as the Nova Scotia variant) is allelic to type C and occurs in descendents of western Nova Scotians.
The biochemical hallmark of NP-C cells is the abnormal accumulation of unesterified cholesterol in lysosomes, which results in delayed homeostatic regulation of both uptake and esterification of low density lipoprotein (LDL) cholesterol (Sokol et al., 1988; Blanchette-Mackie et al., 1988; Pentchev et al., 1994; Pentchev et al., 1987). Accumulation of lysosomal cholesterol in the cells of NP-C sufferers can be detected cytochemically by the cholesterol-specific fluorescent dye, filipin. Normally, endocytosed LDL-derived cholesterol is mobilized from lysosomes to the endoplasmic reticulum for esterification. As a result, there is little free cholesterol accumulation in iysosomes detectable by filipin staining in normal cells. In contrast, in NP-C cells the lysosomal accumulation of the endocytosed LDL-derived free cholesterol results in a specific perinuclear filipin-staining pattern. Biochemically, the NP-C phenotype can most conviently be monitored by LDL-induced cholesterol ester synthesis. Cholesterol ester synthesis is markedly stimulated by LDL in normal cells, but not in NP-C cells.
Two independent murine models having autosomal recessive lysosomal storage defects have been described (Morris et al., 1982; Miyawaki et al., 1982; Sakiyama et al, 1982). The pathological features of these murine mutants are similar to human NP-C (Higashi et al., 1991; Ohno et al., 1992), but, to date, the genetic defect in these mouse lines remains uncharacterized.
If the gene underlying NP-C could be isolated, it could facilitate the detection, diagnosis, and perhaps treatment of the disease. It is the objective of this invention to provide a human cDNA corresponding to the gene for NP-C, as well as the cDNA underlying the NP-C murine models.
The present invention provides, for the first time, an isolated human nucleic acid molecule which is able to correct the cellular defect characteristic of Niemann-Pick type C disease. It is shown that NP-C patients carry mutations in the genomic copies of this nucleic acid. Orthologs of the disclosed nucleic acid molecule from other species are also provided.
More specifically, the invention provides an isolated human cDNA, herein referred to as the human NPC1 cDNA which, when transiently expressed in human cells derived from NP-C patients, is able to correct the abnormal lysosomal cholesterol accumulation that is characteristic of such cells. Also provided by this invention is the nucleotide sequence of this human cDNA molecule, as well as the nucleotide sequences of corresponding cDNAs from mouse, yeast and the worm C. elegans. The amino acid sequences of the proteins encoded by these cDNAs are also provided.
Having provided the nucleotide sequence of the human NPC1 cDNA (as well as the murine ortholog), correspondingly provided are the complementary DNA strands of these cDNA molecules and DNA molecules which hybridize under stringent conditions to these cDNA molecules or their complementary strands. Such hybridizing molecules include DNA molecules differing only by minor sequence changes, including nucleotide substitutions, deletions and additions. Also comprehended by this invention are isolated oligonucleotides comprising at least a segment of the disclosed cDNA molecules or the complementary strands of these molecules, such as oligonucleotides which may be employed as DNA hybridization probes or DNA primers useful in the polymerase chain reactiorn. Hybridizing DNA molecules and variants on the NPC1 cDNAs may readily be created by standard molecular biology techniques.
Through the manipulation of the nucleotide sequences provided by this invention by standard molecular biology techniques, variants of the NPC1 proteins may be made which differ in precise amino acid sequence from the disclosed proteins yet which maintain the basic functional characteristics of the disclosed NPC1 proteins or which are selected to differ in some characteristics from these proteins. Such variants are another aspect of the present invention.
Also provided by the present invention are recombinant DNA vectors comprising the disclosed DNA molecules, and transgenic host cells containing such recombinant vectors.
Having provided the isolated human NPC1 cDNA sequence and the orthologous murine cDNA, also comprehended by this invention are the genomic genes from which these cDNAs are derived.
The present invention also provides methods for using the disclosed cDNAs, the corresponding genomic gene and derivatives thereof, and of the protein, and derivatives thereof, in aspects of diagnosis of NP-C and detection of NP-C carriers. One particular embodiment of the present invention is a method for screening a subject to determine if said subject carries a mutant NPC1 gene. The method comprises detecting the presence of nucleotide differences between the sequence of the subject""s NPC1 gene ORF compared to the NPC1 cDNA sequence disclosed herein, and determining whether any such sequence differences will result in the expression of an aberrant NPC1 gene product in the subject. The step of detecting nucleic acid sequence differences may be performed using several techniques including: hybridization with oligonucleotides (including, for example, the use of high-density oligonucleotide arrays); PCR amplification of the NPC1 gene or a part thereof using oligonucleotide primers; RT-PCR amplification of the NPC1 RNA or a part thereof using oligonucleotide primers, and direct sequencing of the NPC1 gene of the subject""s genome using oligonucleotide primers.
The disclosed sequences will also be useful in the creation and study of mutants in the NPC1 locus, which in turn may yield valuable information about the biochemical pathways underlying the disease, as well as information about cholesterol metabolism.
A further aspect to the present invention is a preparation comprising specific binding agents, such as antibodies, that specifically detect the NPC1 protein. Such specific binding agents may be used in methods for screening a subject to assay for the presence of a mutant NPC1 gene. One exemplary method comprises providing a biological sample of the subject which sample contains cellular proteins and providing an immunoassay for quantitating the level of NPC1 protein in the biological sample.
The foregoing and other features and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. Those skilled in the art will appreciate that the utility of this invention is not limited to the specific experimental modes and materials described herein.